Methods using O6-alkylguanine-DNA alkyltransferases

Abstract

A method using O 6 -alkylguanine-DNA alkyltransferases (AGT) is disclosed for transferring a label from a substrate to a fusion protein comprising the AGT. This allows the detection and/or manipulating of the fusion protein, both in vitro and in vivo, by attaching molecules to the fusion proteins that introduce a new physical or chemical property to the fusion protein. Examples of such molecules are, among others, spectroscopic probes or reporter molecules, affinity tags, molecules generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or molecules suitable for the immobilisation of the fusion protein.

1. A method for transferring a label to a fusion protein, wherein said method comprises contacting a fusion protein comprising a protein fused to an O 6 -alkylguanine-DNA alkyltransferase (AGT) with a substrate having a fluorophore or chromophore label, wherein the AGT transfers the fluorophore or chromophore label from the substrate such that the fluorophore or chromophore label becomes covalently bonded to the fusion protein. 
     
     
       2. A method for transferring a label to a fusion protein, wherein said method comprises contacting a fusion protein comprising a protein fused to an O 6 -alkylguanine-DNA alkyltransferase (AGT) with a labelled substrate represented by the general formula: 
       
         
           
           
               
               
           
         
       
       wherein:
 R 1  is a group accepted by AGT allowing the AGT to transfer the label to the fusion protein; 
 R 2  is a linker group; 
 R 3  is a proton, a β-D-2′-deoxyribosyl, or a β-D-2′-deoxyribosyl that is part of an oligodeoxyribonucleotide; and 
 label represents a fluorophore or chromophore; 
 and wherein the AGT transfers the fluorophore or chromophore label and the fluorophore or chromophore label becomes covalently bonded to the fusion protein. 
 
     
     
       3. A method for transferring a label to a fusion protein, wherein said method comprises contacting a fusion protein comprising a protein of interest fused to an O 6 -alkylguanine-DNA alkyltransferase (AGT) with a benzyl guanine substrate labelled with a fluorophore or chromophore, wherein the AGT transfers the fluorophore or chromophore label and the fluorophore or chromophore label becomes covalently bonded to the fusion protein. 
     
     
       4. The method according to  claim 1 , further comprising detecting the fusion protein using the fluorophore or chromophore label. 
     
     
       5. The method according to  claim 1 , further comprising manipulating the labelled fusion protein using a property introduced by the fluorophore or chromophore label to the fusion protein. 
     
     
       6. The method according to  claim 1 , wherein the O 6 -alkylguanine-DNA alkyltransferase is human O 6 -alkylguanine-DNA alkyltransferase. 
     
     
       7. The method according to  claim 2 , wherein R 1  is a substituted or unsubstituted alkyl chain, a substituted or unsubstituted cycloalkyl group with a ring size between three and ten carbons, a substituted or unsubstituted heterocycle with a ring size between three and ten carbons, or a substituted or unsubstituted aromatic heterocycle with a ring size between three and ten carbons. 
     
     
       8. The method according to  claim 2 , wherein the linker group R 2  is a substituted or unsubstituted alkyl chain or a polyethylene glycol. 
     
     
       9. The method according to  claim 3 , wherein the benzyl guanine substrate is substituted with the fluorophore or chromophore label at position C4 of the benzyl ring. 
     
     
       10. The method according to  claim 3 , wherein the labelled benzyl guanine substrate is represented by the general formula: 
       
         
           
           
               
               
           
         
       
       wherein:
 R 1  is a proton, a β-D-2′-deoxyribosyl or a β-D-2′-deoxyribosyl that is part of an oligodeoxyribonucleotide; 
 R 2  is a linker group; and 
 label represents a fluorophore or chromophore. 
 
     
     
       11. The method according to  claim 4 , which comprises detecting the labelled fusion protein in an in vitro system. 
     
     
       12. The method according to  claim 11 , wherein in the in vitro system, the labelling is carried out in cell extracts or with purified or enriched forms of the fusion protein.